Branched-chain Alcohols Research Articles

Insights into Protein and Amino Acid Metabolism of Thermoanaerobacter mathranii

Few investigations have been carried out into the members of the genus Thermoanerobacter for protein and amino acid metabolism compared to carbohydrates, mostly due to the intense interest in bioethanol and biohydrogen in recent decades. The present study investigates the biotechnological potential of Thermoanaerobacter mathranii (DSM 11426) in terms of its ability to produce high-energy alcohols from amino and fatty acids. End product formation from glucose (in the presence and absence of butyrate) as well as from selected proteins and amino acids were analyzed. T. mathranii did not degrade any of the proteins tested to a large extent but degraded several amino acids, namely serine and the branched-chain amino acids (leucine, isoleucine, valine) when cultivated in the presence of thiosulfate. The main end products from the branched-chain amino acids were a mixture of their corresponding branched-chain fatty acids and alcohols, with the strain producing a concentration of the corresponding branched-chain alcohol between 1.0 and 1.7 mM and 8.2–10.9 mM of the corresponding fatty acid. 13C2-labeled leucine revealed that the strains degraded the amino acid in the presence of thiosulfate, producing 3-methyl-1-butyrate, which was then used as an electron acceptor which led to the accumulation of 3-methyl-1-butanol. The strain is highly ethanologenic, producing more than 1.2 mol of ethanol per mol of glucose degraded. The strain was able to reduce volatile fatty acids during glucose fermentation to their corresponding alcohol, further suggesting this strain may be of greater biotechnological value beyond bioethanol production.

Thermodynamic analysis of adsorption and retention behaviors in normal-phase liquid chromatography

This study investigated the adsorption mechanisms in a normal-phase system using a cyano-based stationary phase as the sorbent. The minor disturbance method was used to measure the adsorption isotherms of acetone and alcohols with various structures. Excluding data in pure n-hexane revealed that the adsorption behaviors on cyano sites were well described by the Langmuir model. The adsorption equilibrium constants, ranging from 8.86 to 11.15 at 25 °C, showed no significant differences across alcohol structures and decreased with increasing temperature. The saturation adsorption concentration decreased with increasing alcohol molecule size, with branched-chain alcohols showing a lower saturation adsorption amount compared to straight-chain alcohols. The standard state adsorption enthalpies and entropies calculated from the equilibrium constants for various alcohols ranged from −29 to −22 kJ/mol and −78 to −55 J/K·mol, respectively, showing enthalpy-entropy compensation. A discrepancy was observed between these adsorption enthalpies and those obtained from the retention factors of alcohols using pure n-hexane as the mobile phase. This discrepancy may result from the affinity energy distribution of the adsorbent. In pure n-hexane, the adsorption behaviors of adsorbates were considerably affected by high-affinity sites. Moreover, acetone and these alcohol molecules were used as solvent modifiers to investigate the relationship between the retention factor, modifier concentration, and temperature for various solutes with distinct functional groups. The retention curves were converted to enthalpic curves using the van't Hoff equation. A theoretical model was proposed to describe the relationship between the van't Hoff enthalpy change and mobile phase composition. The proposed model effectively described the enthalpic curves, indicating that the enthalpy change follows a saturation curve with increasing modifier concentration. This trend is primarily due to competitive adsorption and complexation behaviors between the solute and modifier molecules.

Dual role of triglyceride structures facilitates anti-tumor drug delivery: Both as a self-assembling module and a responsive module

Small molecule prodrugs self-assembled nano-delivery systems with tumor responsive linkages are emerging as an effective platform. However, the heterogeneity of tumor microenvironment may limit the anti-tumor effect of prodrug nanomedicines with a single response module. Here, we chose disulfide bond as the response module and branched chain alcohol as the self-assembly modification module to construct a single-responsive prodrug. We also constructed a double-responsive paclitaxel prodrug combining triglyceride and disulfide bond, taking into account of the highly expressed lipase and glutathione levels in tumor cells. The results showed that the anti-tumor effect of single-responsive branched chain alcohol modified prodrug nanoparticles was inferior to triglyceride prodrug nanoparticles with dual response modules. The triglyceride structure can not only serve as a self-assembly modification module, but also serve as a response module for intelligent drug release in tumor. Such dual roles will facilitate the efficient delivery of small molecule self-assembled prodrugs to tumor sites.

Engineering yeast mitochondrial metabolism for 3-hydroxypropionate production

BackgroundWith unique physiochemical environments in subcellular organelles, there has been growing interest in harnessing yeast organelles for bioproduct synthesis. Among these organelles, the yeast mitochondrion has been found to be an attractive compartment for production of terpenoids and branched-chain alcohols, which could be credited to the abundant supply of acetyl-CoA, ATP and cofactors. In this study we explored the mitochondrial potential for production of 3-hydroxypropionate (3-HP) and performed the cofactor engineering and flux control at the acetyl-CoA node to maximize 3-HP synthesis.ResultsMetabolic modeling suggested that the mitochondrion serves as a more suitable compartment for 3-HP synthesis via the malonyl-CoA pathway than the cytosol, due to the opportunity to obtain a higher maximum yield and a lower oxygen consumption. With the malonyl-CoA reductase (MCR) targeted into the mitochondria, the 3-HP production increased to 0.27 g/L compared with 0.09 g/L with MCR expressed in the cytosol. With enhanced expression of dissected MCR enzymes, the titer reached to 4.42 g/L, comparable to the highest titer achieved in the cytosol so far. Then, the mitochondrial NADPH supply was optimized by overexpressing POS5 and IDP1, which resulted in an increase in the 3-HP titer to 5.11 g/L. Furthermore, with induced expression of an ACC1 mutant in the mitochondria, the final 3-HP production reached 6.16 g/L in shake flask fermentations. The constructed strain was then evaluated in fed-batch fermentations, and produced 71.09 g/L 3-HP with a productivity of 0.71 g/L/h and a yield on glucose of 0.23 g/g.ConclusionsIn this study, the yeast mitochondrion is reported as an attractive compartment for 3-HP production. The final 3-HP titer of 71.09 g/L with a productivity of 0.71 g/L/h was achieved in fed-batch fermentations, representing the highest titer reported for Saccharomyces cerevisiae so far, that demonstrated the potential of recruiting the yeast mitochondria for further development of cell factories.

Tensiometric and Fluorescence Study of Cationic Gemini Surfactant with Some Special Alcohols

Special alcohols have been used as additives to study interfacial properties of cationic Gemini surfactant pentanediyl- 1, 5-bis (dimethyldodecylammonium bromide) (12-5-12). As these branched chain alcohols (in comparison with linear chain alcohols) are playing a measure roll in creating a microemulsion with Gemini surfactants. The surface tension values were measured by using ring detachment method. During the experiments, the ring was cleaned well by heating it in alcohol flame. The critical micelle concentration (cmc) values were obtained from surface tension (γ) versus logCt plots. The γ values decreased continuously and then become constant along a wide concentration range. The point of break, when the constancy of surface tension begins, was taken as the cmc of the system. Calculated Parameters are cmc, Гmax (maximum surface excess concentration), Amin (minimum surface area per molecule), C20 (the concentration of surfactant where the surface tension of the solvent is being reduced by 20 mN.m-1), (free energy of the given air/water interface), and the standard Gibbs energy of adsorption, ΔG­­0ads. An important property of micelle formation is the mean aggregation number which provides direct information about the general size and shape of the aggregates formed by amphiphiles in solution, and how these properties are related to the molecular structure of the amphiphiles. Mixed micellization behavior has been shown by these parameters. The mean aggregation number (Nagg­) of mixed micelles has been obtained by using the steady state fluorescence quenching method. Some other concerned parameters including dielectric constant (D), binding constant (KSV) were calculated in this study by using the ratio of intensity of peaks.

Effects of autochthonous Kluyveromyces lactis and commercial Enterococcus faecium adjunct cultures on the volatile profile and the sensory characteristics of short-ripened acid-curd Cebreiro cheese

Four batches of Cebreiro-type cheese were made in duplicate from pasteurized milk. A control batch was manufactured with only a commercial O-starter. The other three batches were made with the same starter plus: (i) a commercial culture of Enterococcus faecium; (ii) a selected Kluyveromyces lactis adjunct culture used in a cheese-milk pre-ripening step; and (iii) the combination of both adjunct cultures. The cheeses made with the yeast adjunct were characterized by higher values of overall proteolysis, pH and aw, and showed total and lactic acid bacteria (LAB) counts at least 2 log units than the batches made with only LAB. The volatile profiles of the cheeses made with added K. lactis were distinguished by high contents of esters, branched-chain alcohols, fatty acids, acetoin and 2-pnenylethanol. These batches had a more friable and sticky texture, and exhibited differential piquant, yeasty, alcoholic, acetic and fruity flavors. Furthermore, the addition of enterococci seemed to help achieve more desirable sensory characteristics. The batches manufactured with both adjunct cultures were awarded the highest scores for texture preference, flavor intensity, flavor preference, and overall sensory preference. The sensory profiles of the cheeses made with added yeast closely resembled those of traditional ‘good quality’ raw-milk Cebreiro cheese.

Effect of Carbon Sources on the Production of Volatile Organic Compounds by Fusarium verticillioides.

The aim of the present study was to evaluate the effect of different carbon sources on the hydrocarbon-like volatile organic compounds (VOCs) of Fusarium verticillioides strain 7600 through a Principal Component Analysis approach, and to explore their diesel potential by using data from the literature. The fungus was cultivated in GYAM culture medium, and five carbon sources were evaluated: glucose, sucrose, xylose, lactose, and fructose. The VOCs were collected using a close-loop apparatus and identified through GC-MS. The same profile of 81 VOCs was detected with all treatments, but with different relative percentages among carbon sources. The production of branched-chain alkanes (30 compounds) ranged from 25.80% to 38.64%, straight-chain alkanes (12 compounds) from 22.04% to 24.18%, benzene derivatives (12 compounds) from 7.48% to 35.58%, and the biosynthesis of branched-chain alcohols (11 compounds) was from 6.82% to 16.71%, with lower values for the remaining groups of VOCs. Our results show that F. verticillioides has the metabolic potential to synthesize diesel-like VOCs. Further research should include the optimization of culture conditions other than carbon sources to increase the production of certain groups of VOCs.

Escherichia coli coculture for de novo production of esters derived of methyl-branched alcohols and multi-methyl branched fatty acids

BackgroundA broad diversity of natural and non-natural esters have now been made in bacteria, and in other microorganisms, as a result of original metabolic engineering approaches. However, the fact that the properties of these molecules, and therefore their applications, are largely defined by the structural features of the fatty acid and alcohol moieties, has driven a persistent interest in generating novel structures of these chemicals.ResultsIn this research, we engineered Escherichia coli to synthesize de novo esters composed of multi-methyl-branched-chain fatty acids and short branched-chain alcohols (BCA), from glucose and propionate. A coculture engineering strategy was developed to avoid metabolic burden generated by the reconstitution of long heterologous biosynthetic pathways. The cocultures were composed of two independently optimized E. coli strains, one dedicated to efficiently achieve the biosynthesis and release of the BCA, and the other to synthesize the multi methyl-branched fatty acid and the corresponding multi-methyl-branched esters (MBE) as the final products. Response surface methodology, a cost-efficient multivariate statistical technique, was used to empirical model the BCA-derived MBE production landscape of the coculture and to optimize its productivity. Compared with the monoculture strategy, the utilization of the designed coculture improved the BCA-derived MBE production in 45%. Finally, the coculture was scaled up in a high-cell density fed-batch fermentation in a 2 L bioreactor by fine-tuning the inoculation ratio between the two engineered E. coli strains.ConclusionPrevious work revealed that esters containing multiple methyl branches in their molecule present favorable physicochemical properties which are superior to those of linear esters. Here, we have successfully engineered an E. coli strain to broaden the diversity of these molecules by incorporating methyl branches also in the alcohol moiety. The limited production of these esters by a monoculture was considerable improved by a design of a coculture system and its optimization using response surface methodology. The possibility to scale-up this process was confirmed in high-cell density fed-batch fermentations.

Biosensor for branched-chain amino acid metabolism in yeast and applications in isobutanol and isopentanol production

Branched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p from Saccharomyces cerevisiae to develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.

Directed evolution of biofuel-responsive biosensors for automated optimization of branched-chain alcohol biosynthesis

The biosynthesis of short-chain alcohols is a carbon-neutral alternative to petroleum-derived production, but strain screening operations are encumbered by laborious analytics. Here, we built, characterized and applied whole cell biosensors by directed evolution of the transcription factor AlkS for screening microbial strain libraries producing industrially relevant alcohols. A selected AlkS variant was applied for in situ product detection in two screening applications concerning key steps in alcohol production. Further, the biosensor strains enabled the implementation of an automated, robotic platform-based workflow with data clustering, which readily allowed the identification of significantly improved strain variants for isopentanol production.

Quantification of Branched-Chain Alcohol-Based Biofuels and Other Fermentation Metabolites via High-Performance Liquid Chromatography.

As the consequences of climate change become apparent, metabolic engineers and synthetic biologists are exploring sustainable sources for transportation fuels. The design and engineering of microorganisms to produce bio-gasoline and other biofuels from renewable feedstocks can significantly reduce dependence on fossil fuels as well as lower the emissions of greenhouse gases. A significant amount of research over the past two decades has led to the development of microbial strains for the production of advanced fuel compounds. Crucial to these efforts are robust methods to quantify the amount of the biofuel compound being produced as well as the other metabolites that might be present during fermentation. Here, we provide a protocol for the quantification of branched-chain alcohols, specifically isobutanol and isopropanol, using high-performance liquid chromatography (HPLC).

Nitrogen metabolism of branched‐chain alcohols acetates in jujube wine assessed by 13 C‐labeling

Branched-chain alcohols acetates (BCAA) are important aroma components of jujube wine. However, their presence is somewhat difficult to control during fermentation. In this regard, 13C-labeling and Gas Chromatography-Mass Spectrometer (GC-MS) were used to explore the Ehrlich pathway for the formation of BCAA. Initially, branched-chain (BC) amino acids, known to be involved in the production of BCAA, were investigated. BC amino acids, aldehydes, and alcohols were observed to gradually transform, while a small amount of alcohols was esterified to BCAA. Following the addition of 13C-BC amino acids into the jujube pulp, 13C-aldehydes, 13C-alcohols and 13C-BCAA were detected in the wine at 2, 24, and 72 hr, respectively, by mass spectrometric analysis. BC amino acids were observed to be transaminated, decarboxylated to aldehydes and then reduced to alcohols. The alcohols were subsequently esterified with acetyl CoA to form BCAA confirming a potential pathway for flavor regulation and quality improvement of jujube wine. Practical applications Jujube wine, produced by jujube fermentation with Saccharomyces cerevisiae, is a famous alcoholic beverage in Taihang Mountain districts in Hebei with Chinese characteristics. But the development of jujube wine was restricted because its flavor and quality were very unstable. Branched-chain alcohols acetates (BCAA) are the key aroma compounds of jujube wine. Understanding the formation mechanism of BCAA could provide the guide of process optimization and flavor regulation, making it become a unique brandy with Chinese characteristics, recognized, and accepting by the world.

Development of a Simple Colorimetric Assay for Determination of the Isoamyl Alcohol-Producing Strain.

Like other branched-chain higher alcohols used as biofuels, isoamyl alcohol has attracted considerable attention because of its advantages, which include high energy density, low hygroscopicity, and compatibility with the current infrastructure. Previous attempts to increase the microbial production of isoamyl alcohol have yielded great progress, but the existing methods of detecting isoamyl alcohol based on gas chromatography and high-performance liquid chromatography are laborious and time-consuming. In this study, we developed a simple colorimetric assay to determine high isoamyl alcohol-producing strains. The assay was based on isoamyl alcohol oxidase and peroxidase (IAOP assay) and could be performed in microplate with high throughput and had a specific detection range of 0-20mM. Characterization analysis revealed that the developed IAOP assay was highly specific for isoamyl alcohol relative to other branched-chain alcohols. Little interference with the assay was observed from the fermentation media, microorganisms, and fermentation byproducts (e.g., lactic acid, acetic acid). We conclude that the enzyme-based IAOP assay can be used for high-throughput monitoring of strains that produce isoamyl alcohol and could be adjusted to screen for strains that produce many other metabolites.

Effect of biodiesel ester structure optimization on low temperature performance and oxidation stability

The main shortcomings of biodiesel are related to the low-temperature performance and oxidation stability. Hence, this study proposes to investigate the influence of the oleic acid-based esters with various linear and branched chain alcohols on these two properties of biodiesel. The economic impact of biodiesel with various compositions is also evaluated by means of a mathematical model. The results showed that the longer the carbon chain length of the alcohol component, the better the low-temperature performance of the biodiesel. Hence, the cold filter plugging point and solidifying point of methyl oleate were −19 and −24°C, respectively, whereas the cold filter plugging point and solidifying point of amyl oleate were −36 and −41°C, respectively. The more irregular the molecular structure, the better the low-temperature performance of oleic acid-based lipids. For instance, the condensate point and solidifying point of oleic acid-based esters with branched chain alcohols should be lower than oleic acid-based esters with linear chain alcohols. On the other hand, for the linear esters of oleic acid, the oxidation stability is better as the carbon chain is longer. For instance, the induction period of methyl oleate is 0.07h while for amyl oleate, it is 0.36h, which corresponds to an increase of about 5 times. For the branched esters, although they are sensitive to oxidation, the induction period is higher than that of methyl oleate. Finally, the DEA evaluation model was used to analyze the economic aspects of the resulted biodiesel. The results showed that the addition of ethyl oleate to biodiesel significantly improved the low-temperature performance and oxidation stability of biodiesel with good economic benefits.

Sequestration and Elimination of Toxic Aldehydes.

It is well-known that aldehydes resulting from the in vivo oxidation of primary alcohols are toxic. Here, we experimentally demonstrate in rat models that the dipeptide cysteinylglycine (CG), formed in vivo from its oxidized product, cystinyl-bis-glycine (CbG), will sequester acetaldehyde and isoamyl aldehyde, two model aldehydes resulting from the oxidation of ethanol and isoamyl alcohol, respectively, and excrete them in urine as their respective conjugation products with CG. These data suggest that a whole series of toxic aldehydes can be sequestered and detoxified by CG and may prevent the flushing syndrome exhibited by individuals with a defective enzyme that converts acetaldehyde to acetate. The data also suggest the possibility of alleviating the hangover syndrome we believe to be caused by aldehydes, such as isoamyl aldehyde derived from short, branched-chain alcohols, present as congeners in certain alcoholic beverages. The sequestration of other toxic agents, such as cyanide, that can react with CG can also be envisioned.

Effects of mixing between short-chain and branched-chain alcohols in protonated clusters.

The previous analysis of the neat protonated branched-chain alcohol clusters revealed the impact of steric repulsion and dispersion of the bulky alkyl group on the hydrogen-bonded (H-bonded) structures and their temperature-dependence. To further understand the influence of the alkyl groups in H-bonded clusters, we studied the mixing of the two extremes of alcohols, methanol (MeOH) and tert-butyl alcohol (t-BuOH), with an excess proton. Infrared spectroscopy and a structural search of first principles calculations on the size-selected clusters H+(MeOH)m(t-BuOH)t (m + t = 4 and 5) were conducted. Temperature-dependence of the dominant H-bonded structures was explored by the Ar-tagging technique and quantum harmonic superposition approach. By introducing the dispersion-corrected density functional theory methods, it was shown that the effects of dispersion due to the bulky alkyl groups in the mixed clusters cannot be ignored for t≥ 2. The computational results qualitatively depicted the characteristics of the observed IR spectra, but overestimation of the temperature-dependence with dispersion correction was clearly seen due to the unbalanced correction between linear H-bonded structures and compact cyclic ones. These results demonstrate the importance of extensive investigation and benchmarks on different levels of theory, and that a properly sampled structure database is crucial to evaluate theoretical models.

Critical Roles of the Pentose Phosphate Pathway and GLN3 in Isobutanol-Specific Tolerance in Yeast.

Branched-chain alcohols are attractive advanced biofuels; however, their cellular toxicity is an obstacle to engineering microbes to produce them at high titers. We performed genome-wide screens on the Saccharomyces cerevisiae gene deletion library to identify cell systems involved in isobutanol-specific tolerance. Deletion of pentose phosphate pathway genes GND1 or ZWF1 causes hypersensitivity to isobutanol but not to ethanol. By contrast, deletion of GLN3 increases yeast tolerance specifically to branched-chain alcohols. Transcriptomic analyses revealed that isobutanol induces a nitrogen starvation response via GLN3 and GCN4, upregulating amino acid biosynthesis and nitrogen scavenging while downregulating glycolysis, cell wall biogenesis, and membrane lipid biosynthesis. Disruption of this response by deleting GLN3 is enough to enhance tolerance and boost isobutanol production 4.9-fold in engineered strains. This study illustrates how adaptive mechanisms to tolerate stress can lead to toxicity in microbial fermentations for chemical production and how genetic interventions can boost production by evading such mechanisms.

Branched-chain amino acid catabolism of Thermoanaerobacter pseudoethanolicus reveals potential route to branched-chain alcohol formation

The fermentation of branched-chain amino acids (BCAAs) to branched-chain fatty acids (BCFAs) and branched-chain alcohols (BCOHs) is described using Thermoanaerobacter pseudoethanolicus. BCAAs were not degraded without an electron scavenging system but were degraded to a mixture of their BCFA (major) and BCOH (minor) when thiosulfate was added to the culture. Various environmental parameters were investigated using isoleucine as the substrate which ultimately demonstrated that at higher liquid-gas phase ratios the formation of 2-methyl-1-butanol from isoleucine achieved a maximal titer of 3.4mM at a 1:1 liquid-gas ratio suggesting that higher partial pressure of hydrogen influences the BCOH/BCFA ratio but did not increase further with higher L-G phase ratios. Alternately, increasing the thiosulfate concentration decreased the BCOH to BCFA ratio. Kinetic monitoring of BCAA degradation revealed that the formation of BCOHs occurs slowly after the onset of BCFA formation. 13C2-labeled studies of leucine confirmed the production of a mixture of 3-methyl-1-butyrate and 3-methyl-1-butanol, while experiments involving 13C1-labeled 3-methyl-1-butyrate in fermentations containing leucine demonstrated that the carboxylic acid is reduced to the corresponding alcohol. Thus, the role of carboxylic acid reduction is likely of importance in the production of BCOH formation during the degradation of BCAA such as leucine.

Reaction kinetics analysis of branched-chain alkyl esters of palmitic acid and cold flow properties

Preparation of methyl palmitate, isopropyl palmitate, isobutyl palmitate, and isoamyl palmitate was carried out using pyridine n-butyl bisulfate ionic liquid as catalyst in a self-designed reactor to catalyze esterification reaction of palmitic acid with methanol, isopropanol, isobutanol, and isoamyl alcohol, respectively. According to the single-factor experimental results, an orthogonal test of four factors and three levels was carried out for branched-chain alkyl esters of palmitic acid under different reaction time, reaction temperature, and catalyst dosage. Verification test was conducted under the optimal conditions, and the conversion in all the cases was up to 97%. Analysis of the reaction kinetics of methyl palmitate, isopropyl palmitate, isobutyl palmitate, and isoamyl palmitate was carried out by the integral method. Reaction order, frequency factor, activation energy, and reaction kinetic model were determined. Compared to methyl palmitate, the kinematic viscosity of the branched-chain alkyl esters of palmitic acid was slightly higher; however, the solidifying point (SP) and cold filter plugging point (CFPP) decreased with increasing degree of branched-chain. The CFPP reduced by up to 15 °C. Therefore, the use of branched-chain alcohol instead of methanol ester exchange descaling method can effectively reduce the SP and CFPP of biodiesel to improve its cold flow properties.

Catalytic esterification, kinetics, and cold flow properties of isobutyl palmitate

Isobutyl palmitate was synthesized by the esterification of palmitic acid and isobutanol with pyridine n-butyl bisulfate ionic liquid as the catalyst in a self-designed reactor. The kinetics of the esterification and cold flow properties of biodiesel were also studied. The optimal reaction conditions for the esterification are as follows: catalyst dosage, 6%; reaction time, 60 min; molar ratio of isobutanol to palmitic acid, 5:1; and reaction temperature, 100 °C. The verification test was conducted under the optimal conditions, and the esterification rate was up to 98.79%. The kinetics of catalytic esterification was analyzed. The reaction orders was 1.49; the activation energy Ea was 57.38 kJ/mol; the frequency factor A was 2.70 × 107; and the reaction kinetics model was -dcA/dt=2.70×107e-57.38RTcA1.49. The solidifying point (SP), cold filter plugging point (CFPP), and kinematic viscosity of isobutyl palmitate obtained were 3 °C, 7 °C, and 4.93 mm2 s−1, respectively. Compared to methyl palmitate, the SP decreased by six times, and CFPP decreased by 3.28 times. Therefore, the use of branched-chain alcohol instead of methanol ester exchange descaling method can effectively reduce the SP and CFPP of biodiesel to improve cold flow properties of biodiesel.